JPWO2020026733A1 - Fluorescently labeled antibody or antibody fragment - Google Patents

Abstract

An antibody or antibody fragment labeled with a fluorescent dye having the following properties (1) and (2) is provided. (1) The fluorescent dye is quenched in the absence of an antigen and fluorescent in the presence of an antigen. Quenching of the dye is released. (2) The fluorescent dye is contained in an antibody or a compound that binds to an amino acid residue in an antibody fragment.

Description

The present invention relates to fluorescently labeled antibodies or antibody fragments. Since the fluorescently labeled antibody or antibody fragment can detect an antigen by changing the fluorescence intensity, it can be used as a diagnostic agent for a disease or the like.

Sato, one of the present inventors, has recently developed a technique for specifically chemically modifying tyrosine residues in complementarity determining regions while maintaining the antigen recognition ability of an antibody (Non-Patent Document 1). On the other hand, Ueda, one of the inventors of the present invention, is developing a molecule in which a protein having a variable region site structure of an antibody and a fluorescent molecule are conjugated. This molecule is a Turn-ON type fluorescence sensor in which the fluorescence of a fluorescent molecule is quenched in the absence of an antigen, but the quenching is canceled in the presence of an antigen, and is named Q-body (non-patented). Document 2, Patent Document 1, and Patent Document 2).

Japanese Patent No. 5043237 International release 2013/065314

Masayoshi Matsumura, Shinichi Sato, Hiroyuki Nakamura, "Study of reaction conditions for protein functioning by labeling tyrosine residues", Proceedings of the 98th Annual Meeting of the Chemical Society of Japan (2018) R. Abe, H. Ohashi, I. Iijima, M. Ihara, H. Takagi, T. Hohsaka and H. Ueda "" Quenchbodies ": quench-based antibody probes that show antigen-dependent fluorescence" J. Am. Chem. Soc. 133, 17386-17394 (2011).

Q-body is capable of homogeneous antigen detection that does not require solid-phase synthesis or washing steps, and is used in various fields such as clinical diagnosis and environmental analysis. However, a cell-free protein synthesis system was required for efficient Q-body production, and its application to mass synthesis was an issue.

The present invention has been made against such a background, and an object of the present invention is to provide a means for enabling a large-scale supply of Q-body.

By modifying the antibody with a fluorescent dye using the technique of chemically modifying the complementarity determination region of the antibody, the present inventor quenches the fluorescent dye in the absence of the antigen, as in the case of Q-body. , It was found that the quenching of the fluorescent dye is canceled in the presence of the antigen. Further, as the fluorescent dye used at this time, the detection sensitivity of the antigen can be improved by using a fluorescent dye having 4,4-difluoro-4-bora-3a, 4a-diaza-s-indacen (BODIPY) as a basic skeleton. We also found that it would improve. Furthermore, it has been found that the modification of the antibody with a fluorescent dye can also be performed by an electrochemical method. The present invention has been completed based on these findings.

That is, the present invention provides the following [1] to [18].
[1] An antibody or antibody fragment labeled with a fluorescent dye, wherein the fluorescent dye has the following properties (1) and (2).
(1) In the absence of the antigen, the fluorescent dye is quenched, and in the presence of the antigen, the quenching of the fluorescent dye is canceled.
(2) The fluorescent dye is contained in a compound that binds to an amino acid residue in an antibody or antibody fragment.

〔式中、Aは共役環を表し、Lは末端に蛍光色素を有するリンカーを表し（但し、LがA上に存在する場合LはA上の任意の位置に存在してよい。）、R¹は水素原子を表すか、又はA上の任意の位置に存在する１個若しくは２個のアミノ基、アセトアミド基、ヒドロキシ基、アルキル基、若しくはアルコキシ基を表し、R²、R³、R⁴、及びR⁵は、それぞれ水素原子、アルキル基、置換基を有していてもよい芳香族基を表す。〕
で表される化合物であることを特徴とする〔１〕に記載の蛍光標識抗体又は抗体断片。[2] Compounds that bind to amino acid residues in an antibody or antibody fragment are the following general formulas (I) to (VIII).

[In the formula, A represents a conjugated ring, L represents a linker with a fluorescent dye at the end (however, if L is on A, L may be at any position on A), R. ¹ represents a hydrogen atom or represents one or two amino, acetamide, hydroxy, alkyl, or alkoxy groups present at any position on A, R ² , R ³ , R ⁴ , And R ⁵ represent an aromatic group which may have a hydrogen atom, an alkyl group and a substituent, respectively. ]
The fluorescently labeled antibody or antibody fragment according to [1], which is a compound represented by.

[3] The fluorescently labeled antibody or antibody fragment according to [1], wherein the compound that binds to an amino acid residue in the antibody or antibody fragment is a compound that binds to a tyrosine residue in the antibody or antibody fragment. ..

〔式中、Aは共役環を表し、Lは末端に蛍光色素を有するリンカーを表し（但し、LがA上に存在する場合LはA上の任意の位置に存在してよい。）、R¹は水素原子を表すか、又はA上の任意の位置に存在する１個若しくは２個のアミノ基、アセトアミド基、ヒドロキシ基、アルキル基、若しくはアルコキシ基を表し、R²及びR³は、それぞれ水素原子、アルキル基、置換基を有していてもよい芳香族基を表す。〕
で表される化合物であることを特徴とする〔３〕に記載の蛍光標識抗体又は抗体断片。[4] Compounds that bind to tyrosine residues in an antibody or antibody fragment are represented by the following general formulas (I) to (VI).

[In the formula, A represents a conjugated ring, L represents a linker with a fluorescent dye at the end (however, if L is on A, L may be at any position on A), R. ¹ represents a hydrogen atom, or one or two amino groups, acetamide groups, hydroxy groups, alkyl groups, or alkoxy groups present at arbitrary positions on A, and R ² and R ³ represent, respectively. Represents an aromatic group that may have a hydrogen atom, an alkyl group, or a substituent. ]
The fluorescently labeled antibody or antibody fragment according to [3], which is a compound represented by.

〔式中、Lはベンゼン環上の任意の位置に存在し、末端に蛍光色素を有するリンカーを表す。〕
で表される化合物であることを特徴とする〔３〕に記載の蛍光標識抗体又は抗体断片。[5] A compound that binds to a tyrosine residue in an antibody or antibody fragment has the following general formula (Ia).

[In the formula, L represents a linker that exists at an arbitrary position on the benzene ring and has a fluorescent dye at the end. ]
The fluorescently labeled antibody or antibody fragment according to [3], which is a compound represented by.

[6] Any of [1] to [5], wherein the fluorescent dye is a fluorescent dye having 4,4-difluoro-4-bora-3a, 4a-diaza-s-indacen as a basic skeleton. Fluorescently labeled antibody or antibody fragment according to.

[7] The invention according to any one of [1] to [6], wherein the antibody or antibody fragment contains a light chain and a heavy chain, and the fluorescent dye is bound to a variable region of the light chain or the heavy chain. Fluorescently labeled antibody or antibody fragment.

[8] The antibody or antibody fragment is bound to a quencher for the fluorescent dye, and the quencher is bound to a variable region of a chain different from the chain to which the fluorescent dye is bound. The fluorescently labeled antibody or antibody fragment according to [7].

[9] An antibody or antibody fragment is bound to another fluorescent dye that forms a FRET pair with the fluorescent dye, and the other fluorescent dye has a chain different from the chain to which the fluorescent dye is bound. The fluorescently labeled antibody or antibody fragment according to [7], which is bound to a variable region.

[10] The fluorescently labeled antibody or antibody fragment according to any one of [1] to [9], wherein the antibody or antibody fragment is an antibody or antibody fragment against a substance that serves as a marker for a disease.

[11] A diagnostic agent for a disease, which comprises the fluorescently labeled antibody or antibody fragment according to [10].

[12] A method for producing a fluorescently labeled antibody or antibody fragment, which comprises the following steps (1) and (2).
(1) A compound that binds to an amino acid residue in an antibody or antibody fragment and contains a functional group used in a click reaction is reacted with the antibody or antibody fragment to form an amino acid residue in the antibody or antibody fragment. The process of combining,
(2) A step of reacting a fluorescent dye with a functional group used in a click reaction in a compound that binds to an amino acid residue in an antibody or antibody fragment, and binding to this functional group.

〔式中、Aは共役環を表し、Lbは末端にクリック反応に用いられる官能基を有するリンカーを表し（但し、LbがA上に存在する場合LbはA上の任意の位置に存在してよい。）、R¹は水素原子を表すか、又はA上の任意の位置に存在する１個若しくは２個のアミノ基、アセトアミド基、ヒドロキシ基、アルキル基、若しくはアルコキシ基を表し、R²、R³、R⁴、及びR⁵は、それぞれ水素原子、アルキル基、置換基を有していてもよい芳香族基を表す。〕
で表される化合物であることを特徴とする〔１２〕に記載の蛍光標識抗体又は抗体断片の製造方法。[13] Compounds that bind to amino acid residues in an antibody or antibody fragment are represented by the following general formulas (Ib) to (VIIIb).

[In the formula, A represents a conjugated ring and Lb represents a linker having a functional group used for the click reaction at the terminal (however, if Lb is present on A, Lb is present at an arbitrary position on A). Good), R ¹ represents a hydrogen atom, or represents one or two amino, acetamide, hydroxy, alkyl, or alkoxy groups present at any position on A, R ² , R ³ , R ⁴ , and R ⁵ represent aromatic groups that may have hydrogen atoms, alkyl groups, and substituents, respectively. ]
The method for producing a fluorescently labeled antibody or antibody fragment according to [12], which is a compound represented by.

[14] The fluorescently labeled antibody or antibody fragment according to [12], wherein the compound that binds to an amino acid residue in the antibody or antibody fragment is a compound that binds to a tyrosine residue in the antibody or antibody fragment. Manufacturing method.

〔式中、Aは共役環を表し、Lbは末端にクリック反応に用いられる官能基を有するリンカーを表し（但し、LbがA上に存在する場合LbはA上の任意の位置に存在してよい。）、R¹は水素原子を表すか、又はA上の任意の位置に存在する１個若しくは２個のアミノ基、アセトアミド基、ヒドロキシ基、アルキル基、若しくはアルコキシ基を表し、R²及びR³は、それぞれ水素原子、アルキル基、置換基を有していてもよい芳香族基を表す。〕
で表される化合物であることを特徴とする〔１４〕に記載の蛍光標識抗体又は抗体断片の製造方法。[15] Compounds that bind to tyrosine residues in an antibody or antibody fragment are represented by the following general formulas (Ib) to (VIb).

[In the formula, A represents a conjugated ring and Lb represents a linker having a functional group used for the click reaction at the terminal (however, if Lb is present on A, Lb is present at an arbitrary position on A). Good), R ¹ represents a hydrogen atom, or represents one or two amino, acetamide, hydroxy, alkyl, or alkoxy groups present at any position on A, R ² and R ³ represents an aromatic group which may have a hydrogen atom, an alkyl group and a substituent, respectively. ]
The method for producing a fluorescently labeled antibody or antibody fragment according to [14], which is a compound represented by.

〔式中、Lbはベンゼン環上の任意の位置に存在し、末端にクリック反応に用いられる官能基を有するリンカーを表す。〕
で表される化合物であることを特徴とする〔１４〕に記載の蛍光標識抗体又は抗体断片の製造方法。[16] A compound that binds to a tyrosine residue in an antibody or antibody fragment has the following general formula (Iab).

[In the formula, Lb represents a linker that exists at an arbitrary position on the benzene ring and has a functional group used in the click reaction at the end. ]
The method for producing a fluorescently labeled antibody or antibody fragment according to [14], which is a compound represented by.

[17] Any of [12] to [16], wherein the fluorescent dye is a fluorescent dye having 4,4-difluoro-4-bora-3a, 4a-diaza-s-indacen as a basic skeleton. The method for producing a fluorescently labeled antibody or antibody fragment according to the above.

[18] The step (1) is a step of applying a voltage to a solution containing an antibody or an antibody fragment and a compound that binds to an amino acid residue in the antibody or the antibody fragment [12] to [12]. 17] The method for producing a fluorescently labeled antibody or antibody fragment according to any one of.

This specification includes the contents described in the Japanese patent application, Japanese Patent Application No. 2018-142089, and / or drawings which are the basis of the priority of the present application.

The present invention provides novel fluorescently labeled antibodies or antibody fragments. Since the fluorescently labeled antibody or antibody fragment can detect an antigen by changing the fluorescence intensity, it can be used as a diagnostic agent for a disease or the like.

Fluorescence spectrum of _{DBCO-PEG 4-} TAMRA bound to rituximab in the presence or absence of denaturant. Fluorescence spectrum of _{DBCO-PEG 4-} TAMRA not bound to rituximab in the presence or absence of denaturant. Fluorescence spectrum of _{DBCO-PEG 4-} TAMRA bound to rituximab in the presence of cell lysate. As the cell lysate, a lysate of cells expressing the rituximab antigen (left) and a lysate of cells not expressing the rituximab antigen (right) are used. Fluorescence spectrum of DBCO-TAMRA bound to rituximab in the presence of cell lysate. As the cell lysate, a lysate of cells expressing the rituximab antigen (left) and a lysate of cells not expressing the rituximab antigen (right) are used. The figure which shows the structure of the fluorescent dye used in Example 3, the excitation wavelength and the fluorescence wavelength. The figure which represented the electrochemical protein modification method schematically. Fluorescence spectrum of fluorescently labeled rituximab produced by an electrochemical method. The spectra in the presence and absence of the denaturant (denatured) and in the absence of the denaturant (in PBS) are shown.

Hereinafter, the present invention will be described in detail.
(A) Definition In the present invention, the "alkyl group having 1 to 20 carbon atoms" is a linear or branched alkyl group having 1 to 20 carbon atoms, for example, a methyl group, an ethyl group, or n-propyl. Group, iso-propyl group, n-butyl group, iso-butyl group, sec-butyl group, tert-butyl group, pentyl group, iso-pentyl group, neo-pentyl group, hexyl group, iso-hexyl group, heptyl group , Iso-Heptyl group, octyl group, nonyl group, decyl group, undecyl group, dodecyl group, tridecyl group, tetradecyl group, pentadecyl group, hexadecyl group, heptadecyl group, octadecyl group, nonadecil group, icosyl group and the like.

In the present invention, the "alkyl group having 1 to 10 carbon atoms" is a linear or branched alkyl group having 1 or more and 10 or less carbon atoms, and is, for example, a methyl group, an ethyl group, an n-propyl group, or iso-. Propyl group, n-butyl group, iso-butyl group, sec-butyl group, tert-butyl group, pentyl group, iso-pentyl group, neo-pentyl group, hexyl group, iso-hexyl group, heptyl group, iso-heptyl Group, octyl group, nonyl group, decyl group and the like.

In the present invention, the "alkyl group having 1 to 3 carbon atoms" is a linear or branched alkyl group having 1 or more and 3 or less carbon atoms, and is, for example, a methyl group, an ethyl group, an n-propyl group, or iso-. Such as a propyl group.

In the present invention, the "alkoxy group having 1 to 20 carbon atoms" is a linear or branched alkoxy group having 1 to 20 carbon atoms, for example, a methoxy group, an ethoxy group, an n-propoxy group, or iso-. Propoxy group, n-butoxy group, iso-butoxy group, sec-butoxy group, tert-butoxy group, pentyloxy group, iso-pentyloxy group, neo-pentyloxy group, hexyloxy group, iso-hexyloxy group, heptyl Oxy group, iso-heptyloxy group, octyloxy group, nonyloxy group, decyloxy group, undecyloxy group, dodecyloxy group, tridecyloxy group, tetradecyloxy group, pentadecyloxy group, hexadecyloxy group, heptadecyl Oxy group, octadecyloxy group, nonadesyloxy group, icosyloxy group and the like.

In the present invention, the "alkoxy group having 1 to 10 carbon atoms" is a linear or branched alkoxy group having 1 to 10 carbon atoms, for example, a methoxy group, an ethoxy group, an n-propoxy group, or iso-. Propoxy group, n-butoxy group, iso-butoxy group, sec-butoxy group, tert-butoxy group, pentyloxy group, iso-pentyloxy group, neo-pentyloxy group, hexyloxy group, iso-hexyloxy group, heptyl Oxy group, iso-heptyloxy group, octyloxy group, nonyloxy group, decyloxy group and the like.

In the present invention, the "alkoxy group having 1 to 3 carbon atoms" is a linear or branched alkoxy group having 1 or more and 3 or less carbon atoms, and is, for example, a methoxy group, an ethoxy group, an n-propoxy group, or iso-. It is a propoxy group.

In the present invention, the "aromatic group which may have a substituent" means an aromatic group which does not have a substituent or an aromatic group which has at least one substituent. Here, the "aromatic group" refers to a group obtained by removing one hydrogen atom from an aromatic compound, for example, a phenyl group, a 1-naphthyl group, a 2-naphthyl group, a pyridine-2-yl group, and a pyridine-. 3-yl group, pyridine-4-yl group, pyrimidine-2-yl group, pyrimidine-4-yl group, pyrimidine-5-yl group, pyrazine-2-yl group, pyrazine-3-yl group, pyridazine-3 -Il group, pyridazine-4-yl group, furan-2-yl group, furan-3-yl group, thiophen-2-yl group, thiophen-3-yl group, pyrrol-1-yl group, pyrrol-2- Il group, pyrrol-3-yl group, pyrazole-1-yl group, pyrazole-3-yl group, pyrazole-4-yl group, pyrazole-5-yl group, imidazol-1-yl group, imidazol-2-yl group Groups, imidazol-4-yl groups, imidazol-5-yl groups, etc. The substituent can be selected from the group consisting of a methyl group, a fluorine atom, a chlorine atom, a bromine atom, a hydroxy group, a methoxy group and the like. A suitable example of an aromatic group having at least one substituent can be a phenyl group having at least one substituent, and specific examples thereof include a 2-methylphenyl group. 3-Methylphenyl group, 4-Methylphenyl group, 2,3-dimethylphenyl group, 2,4-dimethylphenyl group, 2,5-dimethylphenyl group, 2,6-dimethylphenyl group, 3,4-dimethylphenyl Group, 3,5-dimethylphenyl group, 2-fluorophenyl group, 3-fluorophenyl group, 4-fluorophenyl group, 2,3-difluorophenyl group, 2,4-difluorophenyl group, 2,5-difluorophenyl Group, 2,6-difluorophenyl group, 3,4-difluorophenyl group, 3,5-difluorophenyl group, 2-chlorophenyl group, 3-chlorophenyl group, 4-chlorophenyl group, 2,3-dichlorophenyl group, 2, 4-Dichlorophenyl group, 2,5-dichlorophenyl group, 2,6-dichlorophenyl group, 3,4-dichlorophenyl group, 3,5-dichlorophenyl group, 2-bromophenyl group, 3-bromophenyl group, 4-bromophenyl group , 2,3-Dibromophenyl group, 2,4-dibromophenyl group, 2,5-dibromophenyl group, 2,6-dibromophenyl group, 3,4-dibromophenyl group, 3,5-dibromophenyl group, 2 -Hydroxyphenyl group, 3-hydroxyphenyl group, 4-hydroxyphenyl group, 2-methoxyphenyl group, 3-methoxyphenyl group, 4-methoxyphenyl group and the like can be mentioned.

In the present invention, the "functional group used in the click reaction" is, for example, an azide group or an ethynyl group.

In the present invention, the "conjugated ring" means a ring having a conjugated double bond. The conjugated ring may be an aromatic ring or a non-aromatic ring. Further, the conjugated ring may be a ring composed of only carbon atoms or a heterocycle containing atoms other than carbon, for example, atoms such as nitrogen, oxygen and sulfur. Specific examples of the conjugated ring include a 6-membered ring such as a benzene ring, a 1,3-cyclohexadiene ring, a pyridine ring, a pyrimidine ring, a pyrazine ring, a pyridazine ring, and a triazine ring, a cyclopentadiene ring, a furan ring, a thiophene ring, and a pyrazole. Examples thereof include a 5-membered ring such as a ring, a pyrazole ring, and an imidazole ring.

(B) Fluorescent-labeled antibody or antibody fragment The fluorescent-labeled antibody or antibody fragment of the present invention is labeled with a fluorescent dye, and this fluorescent dye is quenched in the absence of an antigen, similar to the fluorescent dye in Q-body. , The quenching is canceled in the presence of the antibody. Since such quenching and its cancellation are due to the interaction with the tryptophan residues present in the heavy chain variable region and the light chain variable region of the antibody, the fluorescent dye can interact with these tryptophan residues. It is preferably present in position. Specifically, the fluorescent dye preferably binds to the variable region of the heavy chain or light chain in the antibody or antibody fragment via a linker, and determines the complementarity of the heavy chain or light chain in the antibody or antibody fragment. It is more preferable to combine with the region. Since the complementarity determining regions are involved in the recognition ability of antigens, it has been thought that the antigen recognition ability is lost when a fluorescent dye is bound to such regions. We have confirmed that cognitive ability is not lost.

Further, the fluorescent dye is contained in a compound that binds to an amino acid residue in the antibody or antibody fragment, and when such a compound binds to an amino acid residue in the antibody or antibody fragment, the antibody or antibody fragment becomes a fluorescent dye. Marked with.

Examples of the compound that binds to the amino acid residue in the antibody or antibody fragment include compounds represented by the following general formulas (I) to (VIII).

In the general formulas (I), (II), (VII), and (VIII), A represents a conjugated ring. A may be a conjugated ring, but in the general formulas (I), (II), and (VII), it is preferably a benzene ring, and in the general formula (VIII), it is preferably a pyridine ring.

In general formulas (I) and (II), R ¹ represents a hydrogen atom, or one or two amino groups, acetamide groups, hydroxy groups, alkyl groups, or alkoxy groups present at arbitrary positions on A. Represents. Here, the number of carbon atoms of the alkyl group and the alkoxy group is not particularly limited, but the number of carbon atoms is preferably 1 to 20, more preferably 1 to 10 carbon atoms, and 1 to 3 carbon atoms. Is particularly preferred. R ¹ may be the above-mentioned group, but is preferably a hydrogen atom or one amino group, an acetamide group, or a methoxy group, and more preferably a hydrogen atom or one methoxy group. .. When two of the above substituents are present, each substituent may be the same or different. The substituent may be present at an arbitrary position on the conjugated ring, but is preferably present at a position away from the adjacent heterocycle for ease of synthesis and the like.

In the general formulas (I), (II), (III), (IV), (V), (VII), and (VIII), R ² may have a hydrogen atom, an alkyl group, and a substituent. Represents a family group. Here, the number of carbon atoms of the alkyl group is not particularly limited, but the number of carbon atoms is preferably 1 to 20, more preferably 1 to 10 carbon atoms, and particularly preferably 1 to 3 carbon atoms. preferable. R ² may be the above-mentioned group, but in the general formula (I), it is preferably a hydrogen atom, a methyl group, or a phenyl group, more preferably a methyl group, and in the general formula (II). Is preferably a hydrogen atom or a methyl group, more preferably a methyl group, and in the general formula (III), a hydrogen atom, a methyl group, a phenyl group, a phenyl group having a methoxy group, and a phenyl group having a fluorine atom. , More preferably a methyl group, preferably a hydrogen atom or a methyl group in the general formulas (IV), (V) and (VII), and a hydrogen atom in the general formula (VIII). Is preferable.

In the general formulas (I), (II), (III), (IV), (V), (VII), and (VIII), R ³ may have a hydrogen atom, an alkyl group, and a substituent. Represents a family group. Here, the number of carbon atoms of the alkyl group is not particularly limited, but the number of carbon atoms is preferably 1 to 20, more preferably 1 to 10 carbon atoms, and particularly preferably 1 to 3 carbon atoms. preferable. R ³ may be the above-mentioned group, but is preferably a hydrogen atom in the general formulas (I), (II), and (III), and is a hydrogen atom in the general formulas (IV) and (V). Alternatively, it is preferably a methyl group, and in the general formulas (VII) and (VIII), it is preferably a hydrogen atom.

In the general formulas (VII) and (VIII), R ⁴ represents an aromatic group which may have a hydrogen atom, an alkyl group and a substituent. Here, the number of carbon atoms of the alkyl group is not particularly limited, but the number of carbon atoms is preferably 1 to 20, more preferably 1 to 10 carbon atoms, and particularly preferably 1 to 3 carbon atoms. preferable. R ⁴ may be any of the above-mentioned groups, but in the general formula (VII), it is preferably a hydrogen atom or a methyl group, and in the general formula (VIII), it is preferably a hydrogen atom.

In general formula (VII), R ⁵ represents an aromatic group which may have a hydrogen atom, an alkyl group and a substituent. Here, the number of carbon atoms of the alkyl group is not particularly limited, but the number of carbon atoms is preferably 1 to 20, more preferably 1 to 10 carbon atoms, and particularly preferably 1 to 3 carbon atoms. preferable. R ⁵ may be any of the groups described above, but is preferably a hydrogen atom.

In the general formulas (I), (II), (III), (IV), (V), (VI), (VII), and (VIII), L represents a linker having a fluorescent dye at the terminal. Further, when L exists on A, L may exist at an arbitrary position on A. The linker may have any structure as long as it does not lose the function of the fluorescent dye, but is preferably an alkylene group. However, one or more -CH _2- of the alkylene group may be substituted with -O-, -S-, -NH-, or -CO-. Further, in the present invention, since the fluorescent dye is usually bound by using the click reaction, 1H-1,2,3-triazole-1,4- caused by the click reaction (reaction between the azide group and the ethynyl group). The diyl group may be substituted _{with -CH 2-.} The number of carbon atoms of the alkylene group is not particularly limited, but is preferably 5 to 20, more preferably 5 to 15. _{Even when -CH 2-} in the alkylene group is replaced with another group, the other group is also included in the above-mentioned "carbon number of alkylene group" as having one carbon.

上記の化合物におけるLは一般式(I)〜(VIII)におけるLと同じ意味であり、Xは酸素原子又は硫黄原子を表し、RはXが酸素原子を表す場合は水素原子又はメチル基を表し、Xが硫黄原子を表す場合は水素原子を表す。The compound that binds to an amino acid residue in an antibody or antibody fragment is not limited to the compounds represented by the above general formulas (I) to (VIII), and includes, for example, the following compounds.

L in the above compound has the same meaning as L in the general formulas (I) to (VIII), X represents an oxygen atom or a sulfur atom, and R represents a hydrogen atom or a methyl group when X represents an oxygen atom. , When X represents a sulfur atom, it represents a hydrogen atom.

Among the compounds that bind to amino acid residues in an antibody or antibody fragment, compounds that bind to tyrosine residues in an antibody or antibody fragment are preferred. All the compounds represented by the general formulas (I) to (VI) can bind to tyrosine residues. That is, the compound represented by the general formula (I) (Japanese Patent Laid-Open No. 2016-108266, WO2017 / 061288) binds to the tyrosine residue, and the compound represented by the general formulas (II) and (III) (S. Sato et al., Chem. Commun. 54, 5871-5874 (2018)) also binds to tyrosine residues. In addition, the compound represented by the general formula (IV) (S. Sato et al., Angew. Chem. Int. Ed. 52, 8681-8684 (2013)) and the compound represented by the general formula (V) (KL). Seim et al., J. Am. Chem. Soc. 133, 16970-16976 (2011)), a compound represented by the general formula (VI) (JS Rees et al., Mol Cell Proteomics. 14 (11): 2848 -2856 (2015)) binds to electron-rich amino acid residues such as tyrosine residues, tryptophan residues, free cysteine residues, and histidine residues.

As described above, the compounds represented by the general formulas (I) to (III) specifically bind to the tyrosine residue in the antibody or antibody fragment and are represented by the general formulas (VI) to (VI). The compound can also bind to tyrosine residues in the antibody or antibody fragment. Since there are many surface-exposed tyrosine residues in the complementarity determining regions of an antibody, the complementarity determination of an antibody or antibody fragment can be determined by using the compounds represented by the general formulas (I) to (VI). The fluorescent dye can be bound to the region in a site-specific manner.

で表される化合物を使用するのが好ましい。ここで、Lはベンゼン環上の任意の位置に存在してよいが、フタル酸ヒドラジド上の６位又は７位に存在することが好ましい。また、一般式(Ia)で表される化合物は、一種類の化合物だけ使用してもよいが、二種類以上の化合物の混合物を使用してもよい。例えば、Lが６位に存在する化合物とLが７位に存在する化合物の混合物を使用してもよい。As the compound that binds to the tyrosine residue in the antibody or antibody fragment, any of the compounds represented by the general formulas (I) to (VI) may be used, but the compound represented by the general formula (I) may be used. It is preferable to use. Further, among the compounds represented by the general formula (I), the following general formula (Ia)

It is preferable to use the compound represented by. Here, L may be present at any position on the benzene ring, but is preferably present at the 6th or 7th position on the phthalic acid hydrazide. Further, as the compound represented by the general formula (Ia), only one kind of compound may be used, or a mixture of two or more kinds of compounds may be used. For example, a mixture of a compound in which L is at the 6-position and a compound in which L is at the 7-position may be used.

As the fluorescent dye, the fluorescent dye used in the conventional Q-body, for example, the fluorescent dye described in the specification of WO2013 / 065314 can be used. Specifically, rhodamine, coumarin, Cy, EvoBlue, oxazine, Carbopyronin, naphthalene, biphenyl, anthracene, phenenthrene, pyrene, carbazole, BODIPY (4,4-difluoro-4-bora-3a, 4a-diaza-s-indacene) ) And the like as a basic skeleton, and derivatives of the fluorescent dye can be exemplified. Specific examples of the fluorescent dye include CR110: carboxylrhodamine 110: Rhodamine Green (trade name), TAMRA: carbocytetremethlrhodamine: TMR, Carboxyrhodamine 6G: CR6G, ATTO655 (trade name), BODIPY FL (trade name): 4,4-difluoro- 5,7-dimethyl-4-bora-3a, 4a-diaza-s-indancene-3-propionic acid, BODIPY 493/503 (Trademark): 4,4-difluoro-1,3,5,7-tetramethyl- 4-bora-3a, 4a-diaza-s-indancene-8-propionicacid, BODIPY R6G (trade name): 4,4-difluoro-5- (4-phenyl-1,3-butadienyl) -4-bora-3a , 4a-diaza-s-indancene-3-propionic acid, BODIPY 558/568 (trade name): 4,4-difluoro-5- (2-thienyl) -4-bora-3a, 4a-diaza-s-indancene -3-propionic acid, BODIPY 564/570 (trademark): 4,4-difluoro-5-styryl-4-bora-3a, 4a-diaza-s-indancene-3-propionic acid, BODIPY 576/589 (trademark) Name): 4,4-difluoro-5- (2-pyrrolyl) -4-bora-3a, 4a-diaza-s-indancene-3-propionic acid, BODIPY 581/591 (Trademark): 4,4-difluoro -5- (4-phenyl-1, 3-butadienyl) -4-bora-3a, 4a-diaza-s-indancene-3-propionic acid, BODIPY TMR (trademark), Cy3 (trademark), Cy3B (trademark) Name), Cy3.5 (trademark name), Cy5 (trademark name), Cy5.5 (trademark name), EvoBlue10 (trademark name), EvoBlue30 (trademark name), MR121, ATTO 390 (trademark name), ATTO 425 (trademark name) Name), ATTO 465 (trade name), ATTO 488 (trade name), ATTO 495 (trade name), ATTO 520 (Trademark name), ATTO 532 (Trademark name), ATTO Rho6G (Trademark name), ATTO 550 (Trademark name), ATTO 565 (Trademark name), ATTO Rho3B (Trademark name), ATTO Rho11 (Trademark name), ATTO Rho12 (Trademark name) Trademark name), ATTO Thio12 (trademark name), ATTO 610 (trademark name), ATTO 611X (trademark name), ATTO 620 (trademark name), ATTO Rho14 (trademark name), ATTO 633 (trademark name), ATTO 647 (trademark name) Name), ATTO 647N (trademark), ATTO 655 (trademark), ATTO Oxa12 (trademark), ATTO 700 (trademark), ATTO 725 (trademark), ATTO 740 (trademark), Alexa Fluor 350 (trademark) Name), Alexa Fluor 405 (Trademark), Alexa Fluor 430 (Trademark), Alexa Fluor 488 (Trademark), Alexa Fluor 532 (Trademark), Alexa Fluor 546 (Trademark), Alexa Fluor 555 (Trademark) , Alexa Fluor 568 (Trademark), Alexa Fluor 594 (Trademark), Alexa Fluor 633 (Trademark), Alexa Fluor 647 (Trademark), Alexa Fluor 680 (Trademark), Alexa Fluor 700 (Trademark), Alexa Fluor 750 (trademark), Alexa Fluor 790 (trademark), Rhodamine Red-X (trademark), Texas Red-X (trademark), 5 (6) -TAMRA-X (trademark), 5TAMRA (trademark) ), SFX (trade name) can be mentioned. Among these, CR110 and TAMRA, which are rhodamine-based fluorescent dyes, ATTO655, which is an oxazine-based fluorescent dye, BODIPY FL, BODIPY R6G, BODIPY 558/568, BODIPY 581/591, and BODIPY TMR, which are BODIPY-based fluorescent dyes, are particularly suitable. It can be exemplified as a fluorescent dye.

The fluorescent dye is usually bound to a compound represented by the general formulas (I) to (VI) via a group used for a click reaction such as an azide group. Therefore, it is preferable that the fluorescent dye has a structure that binds to an azide group, for example, an ethynyl group or a cycloalkyne. More specifically, the fluorescent dye preferably has dibenzylcyclooctyne (DBCO) or the like. The fluorescent dye may be bound to a compound such as DBCO via a linker, or may be bound to a compound such as DBCO without a linker.

The antibody to be used is not particularly limited, and for example, rituximab, which is an anti-CD20 antibody, trastuzumab, which is an anti-HER2 antibody, and the like can be used. In addition, although Q-body targets small molecule compounds (WO2013 / 065314), the fluorescently labeled antibody or antibody fragment of the present invention also targets small molecule compounds, so antibodies against small molecule compounds are used. You may. The low molecular weight compounds referred to here are, for example, stimulants and drugs such as amphetamine, methanephetamine, morphine, heroine, and codeine, aflatoxin, sterigmatocystin, neosolaniol, nivalenol, fumonicin, okratoxin, and endocrine disrupting toxins. Mold poisons such as mold toxins, sex hormones such as testosterone and estradiol, additives illegally used in feeds such as clembuterol and lactopamine, harmful substances such as PCB, gosipole, histamine, benzpyrene, melamine, acrylamide, dioxin, acetamiprid, imidacloprid, chlorphenapir , Malathion, carbalyl, clothianidin, triflumizole, chlorotalonyl, spinosad, lanate, metamidphos, residual pesticides such as chlorpyrifos, environmental hormones such as bisphenol A, etc. Furthermore, when the fluorescently labeled antibody or antibody fragment of the present invention is used as a diagnostic agent for a disease, an antibody against a substance that serves as a marker for the disease can be used.

As the antibody fragment, the above-mentioned antibody fragment can be used. Further, the antibody fragment may be any fragment as long as the fluorescent dye bound to the fragment is quenched in the absence of the antigen and the quenching is canceled in the presence of the antigen, as in the case of the full-length antibody. Since it is usually necessary for the antibody fragment to have a light chain variable region and a heavy chain variable region in order for the fluorescent dye to exhibit such a change, it is preferable that the antibody fragment contains these two regions. Specific examples of the antibody fragment containing such a light chain variable region and a heavy chain variable region include a Fab fragment, an F (ab') ₂ fragment, and a scFv (single-chain variable fragment).

Quenching or FRET (Fluorescence Resonance Energy Transfer) for fluorescent dyes can also be used to increase the decrease in fluorescence intensity when quenching and the increase in fluorescence intensity when quenching the fluorescent dye (R. Abe et al). ., Scientific Reports 4, 4640 (2014), WO2013 / 065314).

As a method of utilizing the quencher, a method of binding the quencher to a variable region of a chain (light chain or heavy chain) to which the fluorescent dye is bound and another chain can be exemplified. By binding the quencher to such a position, the quencher approaches the fluorescent dye in the absence of the antigen and exhibits a quenching effect, but in the presence of the antigen, the quencher separates from the fluorescent dye and does not exhibit the quenching effect. .. As a result, the decrease in fluorescence intensity when quenching the fluorescent dye and the increase in fluorescence intensity when quenching can be further increased.

The quencher used is not particularly limited as long as it exhibits a quenching effect on the fluorescent dye. Examples of such a quencher include an extinguishing dye having NBD: 7-nitrobenzofurazan, DABCYL, BHQ, ATTO, QXL, QSY, Cy, Lowa Black, IRDYE and the like as a basic skeleton and a derivative of the extinguishing dye. , Specifically, NBD, DABCYL, BHQ-1 (trademark name), BHQ-2 (trademark name), BHQ-3 (trademark name), ATTO540Q (trademark name), ATTO580Q (trademark name), ATTO612Q (trademark name) ), QXL490 (trademark name), QXL520 (trademark name), QXL570 (trademark name), QXL610 (trademark name), QXL670 (trademark name), QXL680 (trademark name), QSY-35 (trademark name), QSY-7 ( Trademark name), QSY-9 (trademark name), QSY-21 (trademark name), Cy5Q (trademark name), Cy7Q (trademark name), Lowa Black FQ (trademark name), Lowa Black RQ (trademark name), IRDYE QC -1 (trade name) can be mentioned, but among these, NBD is preferable. In addition, the combination of the fluorescent dye and the quencher in the fluorescently labeled antibody or antibody fragment of the present invention effectively quenches the fluorescent dye in the absence of the antigen and does not inhibit the emission of the fluorescent dye in the presence of the antigen. The combination can be appropriately selected, and the combination of the fluorescent dyes TAMRA and NBD can be exemplified.

Binding of the quencher to the antibody or antibody fragment modifies the method of utilizing the compound (for example, the compound represented by the general formulas (I) to (VIII)) that binds to the amino acid residue in the antibody or antibody fragment described above. That is, the method using a compound in which the fluorescent dye contained in the compound that binds to the amino acid residue in the antibody or antibody fragment is replaced with a quencher may be used, but other methods may be used.

Examples of the method using FRET include a method of binding another fluorescent dye that forms a FRET pair with the fluorescent dye to a variable region of the chain (light chain or heavy chain) to which the fluorescent dye is bound and another chain. can.

By binding another fluorescent dye that forms a FRET pair at such a position, the other fluorescent dye is in close proximity to the fluorescent dye in the absence of the antigen, resulting in FRET, but in the presence of the antigen, the other fluorescent dye is It separates from the fluorescent dye and does not cause FRET. As a result, the decrease in fluorescence intensity when quenching the fluorescent dye and the increase in fluorescence intensity when quenching can be further increased.

Examples of the fluorescent dye forming such a FRET pair include a combination of CR110 and TAMRA.

The binding of another fluorescent dye to the antibody or antibody fragment is a method using a compound (for example, a compound represented by the general formulas (I) to (VIII)) that binds to an amino acid residue in the antibody or antibody fragment described above. It may be done by, but it may be done by another method.

The fluorescently labeled antibody or antibody fragment of the present invention has the following advantages.
1) Since tyrosine residues selectively present in the complementarity determining regions of antibodies can be efficiently (almost 100%) labeled, a Q-body-ized preparation such as an antibody drug has been established. It is possible to do.
2) It can be synthesized by a method based on a chemical reaction and does not require a genetic engineering method, so that it can be synthesized in large quantities.
3) It becomes possible to make a polyclonal antibody into a Q-body, which was difficult in the past.

(C) Diagnostic Agent The diagnostic agent for a disease of the present invention contains the fluorescently labeled antibody or antibody fragment of the present invention described above, and the antibody or antibody fragment is an antibody or antibody fragment against a substance that serves as a marker for the disease. It is characterized by that.

Examples of the antibody against the substance that is a marker of the disease include the above-mentioned rituximab and trastuzumab. Rituximab is an antibody against CD20, a marker of malignant lymphoma, and trastuzumab is an antibody against HER2, a marker of breast, esophageal, and gastric cancer. Many other antibodies to disease marker substances are known, and most of them are commercially available. Such known antibodies or commercially available antibodies can be used in the diagnostic agent for the disease of the present invention. Specific examples of antibodies against substances that are markers of diseases include antibodies against EGFR, which is a marker for lung cancer, antibodies against α-fetoprotein (AFP), which is a marker for liver cancer, and markers for thyroid medullary cancer. Antibodies to calcitonin, antibodies to CA15-3, which is a marker for breast cancer, antibodies to CA19-9, which is a marker for bile duct cancer, antibodies to CD45, which is a marker for leukemia, and antibodies to Myo D1 which is a marker for horizontal print myoma. , Antibodies to PLAP, which is a marker for fetal cancer, antibodies to PSA, which is a marker for prostate cancer, antibodies to TTF1 which is a marker for thyroid cancer, antibodies to amyloid β and tau protein, which are markers for Alzheimer's disease, etc. Can be mentioned.

The diagnostic agent for a disease of the present invention can be used as an in vitro diagnostic agent for examining a marker substance in a sample collected from an organism such as human (for example, blood, cerebrospinal fluid, saliva, urine, feces, skin, etc.). It can also be administered to an organism such as a human and used as an in-vivo diagnostic agent for examining a marker substance present in an organ, tissue, or cell in the organism.

(D) Method for producing fluorescently labeled antibody or antibody fragment The method for producing a fluorescently labeled antibody or antibody fragment of the present invention includes the following steps (1) and (2).

In step (1), a compound that binds to an amino acid residue in an antibody or antibody fragment and contains a functional group used in a click reaction is reacted with the antibody or antibody fragment to cause an amino acid in the antibody or antibody fragment. Combine with residues.

Examples of the compound that binds to the amino acid residue in the antibody or antibody fragment include compounds represented by the following general formulas (Ib) to (VIIIb).

Formula (Ib) A in ^{~ (VIIIb), R 1,} R 2, R 3, R 4, and R ^5, A in formula (I) ~ (VIII), R 1, R 2, R 3, It has the same meaning as R ⁴ and R ^5.

In the general formulas (Ib) to (VIIIb), Lb represents a linker having a functional group used in the click reaction at the terminal. Further, when Lb exists on A, Lb may exist at an arbitrary position on A. The linker may have any structure as long as it does not lose the function of the functional group used in the click reaction, but is preferably an alkylene group. However, one or more -CH _2- of the alkylene group may be substituted with -O-, -S-, -NH-, or -CO-. The number of carbon atoms of the alkylene group is not particularly limited, but is preferably 5 to 20, more preferably 5 to 15. _{Even when -CH 2-} in the alkylene group is replaced with another group, the other group is also included in the above-mentioned "carbon number of alkylene group" as having one carbon.

上記の化合物におけるLbは一般式(Ib)〜(VIIIb)におけるLbと同じ意味であり、Xは酸素原子又は硫黄原子を表し、RはXが酸素原子を表す場合は水素原子又はメチル基を表し、Xが硫黄原子を表す場合は水素原子を表す。The compound that binds to an amino acid residue in an antibody or antibody fragment is not limited to the compounds represented by the above general formulas (Ib) to (VIIIb), and includes, for example, the following compounds.

Lb in the above compound has the same meaning as Lb in the general formulas (Ib) to (VIIIb), X represents an oxygen atom or a sulfur atom, and R represents a hydrogen atom or a methyl group when X represents an oxygen atom. , When X represents a sulfur atom, it represents a hydrogen atom.

Among the compounds that bind to amino acid residues in an antibody or antibody fragment, compounds that bind to tyrosine residues in an antibody or antibody fragment are preferred. Examples of such a compound include compounds represented by the general formulas (Ib) to (VIb).

で表される化合物を使用するのが好ましい。ここで、Lbはベンゼン環上の任意の位置に存在してよいが、フタル酸ヒドラジド上の６位又は７位に存在することが好ましい。また、一般式(Iab)で表される化合物は、一種類の化合物だけ使用してもよいが、二種類以上の化合物の混合物を使用してもよい。例えば、Lbが６位に存在する化合物とLbが７位に存在する化合物の混合物を使用してもよい。Among the compounds represented by the general formulas (Ib) to (VIb), it is preferable to use the compound represented by the general formula (Ib). In addition, among the compounds represented by the general formula (Ib), the following general formula (Iab)

It is preferable to use the compound represented by. Here, Lb may be present at an arbitrary position on the benzene ring, but is preferably present at the 6th or 7th position on the phthalic acid hydrazide. Further, as the compound represented by the general formula (Iab), only one kind of compound may be used, or a mixture of two or more kinds of compounds may be used. For example, a mixture of a compound having Lb at the 6-position and a compound having Lb at the 7-position may be used.

A compound that binds to an amino acid residue in an antibody or antibody fragment and the antibody or antibody fragment are usually reacted in the presence of a catalyst such as HRP (horseradish peroxidase) and an oxidizing agent such as hydrogen peroxide. The reaction may be performed electrochemically without using a catalyst or an oxidizing agent.

(a) Reaction using a catalyst and an oxidizing agent The amount of the compound that binds to the amino acid residue in the antibody or antibody fragment used in this reaction is not particularly limited, but the compound represented by the general formula (Ib) is used. In the case, it is usually 1 to 1000 mol, preferably 10 to 100 mol, with respect to 1 mol of the antibody or antibody fragment.

The solvent used for this reaction is not particularly limited as long as it does not inhibit the reaction, and for example, water, acetonitrile, methanol, ethanol, dimethyl sulfoxide and the like can be used.

The pH at the time of reaction is not particularly limited, but is usually 3 to 11, preferably 6 to 8.

The reaction temperature is not particularly limited, but is usually 4 to 50 ° C, preferably 10 to 30 ° C.

The reaction time is not particularly limited, but is usually 5 to 240 minutes, preferably 30 to 60 minutes.

When HRP is used as a catalyst, the amount thereof is not particularly limited, but is usually 0.001 to 10 mol, preferably 0.001 to 0.005 mol, per 1 mol of the antibody or antibody fragment.

When hydrogen peroxide is used as an oxidizing agent, the amount thereof is not particularly limited, but is usually 1 to 100 mol, preferably 1 to 10 mol, per 1 mol of the antibody or antibody fragment.

(b) Electrochemical reaction A compound that binds to an amino acid residue in an antibody or antibody fragment and an antibody or antibody fragment may be electrochemically reacted by applying a voltage to a solution containing both.

The solvent used is not particularly limited as long as it is conductive and does not adversely affect the antibody or antibody fragment. For example, an electrolyte solution such as Tris-HCl buffer or phosphate buffer (eg, potassium phosphate buffer) can be used as the solvent. The pH of the solution is not particularly limited, but is preferably 5 to 9, more preferably 6 to 8.

The concentration of the compound that binds to the amino acid residue in the antibody or antibody fragment in the solution is appropriately set depending on the type of the compound used, but when the compound represented by the general formula (Ib) is used, it is preferable. , 50-1000 μM, more preferably 100-500 μM.

The concentration of the antibody or antibody fragment in the solution is not particularly limited, but is preferably 0.1 to 100 μM, and more preferably 1 to 10 μM.

The voltage can be applied, for example, by putting a solution in an electrolytic cell, inserting an electrode into the solution, and applying a voltage from the outside between the electrodes. Such a voltage can be applied using a commercially available bulk electrolysis cell or the like. The electrode may be the same as that used in a general electrochemical reaction, and for example, a carbon electrode, a platinum electrode, a silver electrode, a copper electrode, or the like can be used as the electrode. As the carbon electrode, it is preferable to use a porous carbon electrode having a large surface area.

The electrolysis method may be either a constant current method or a constant voltage method, but the constant voltage method is preferable. In the case of the constant voltage method, it is preferable to use a commercially available potentiostat.

When a voltage is applied by a constant voltage method, the voltage is not particularly limited, but is preferably 0.1 to 5.0 V, and more preferably 0.1 to 2.0 V.

The voltage application time (cumulative time) is not particularly limited, but is preferably 1 second to 120 minutes, and more preferably 1 to 60 minutes.

After the reaction of (a) or (b) above is completed, substances other than the target product (antibody or antibody fragment bound to the compounds represented by the general formulas (Ib) to (VIIIb)) are chromatographed. It may be removed by using or the like.

In step (2), the fluorescent dye is reacted with a functional group used in the click reaction in the compounds represented by the general formulas (Ib) to (VIb), and is bonded to this functional group.

The step (2) may be performed continuously or discontinuously with the step (1).

The reaction of step (2) is a reaction well known as a click reaction, and those skilled in the art can appropriately set the conditions.
As the fluorescent dye, the same one as described in "(B) Fluorescently labeled antibody or antibody fragment" can be used.

The amount of the fluorescent dye used in this reaction is not particularly limited, but when DBCO-TAMRA is used as the fluorescent dye, it is usually 1 to 100 mol, preferably 1 to 1 to 1 mol per 1 mol of the antibody or antibody fragment. It is 10 mol.

The solvent used for this reaction is not particularly limited as long as it does not inhibit the reaction, and for example, water, acetonitrile, methanol, ethanol, dimethyl sulfoxide and the like can be used.

The pH at the time of reaction is not particularly limited, but is usually 3 to 11, preferably 6 to 8.

The reaction temperature is not particularly limited, but is usually 4 to 50 ° C, preferably 25 to 40 ° C.

The reaction time is not particularly limited, but is usually 5 to 720 minutes, preferably 5 to 60 minutes.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

[Example 1] Release of dimming with a denaturing agent Rituximab (Chugai Pharmaceutical Co., Ltd.), which is an anti-human CD20 antibody, was dissolved in TBS Tris Buffered Saline (100 mM Tris-HCl, 150 mM NaCl pH 7.4) at a concentration of 5 μM, and HRP and NMe Luminol-N ₃ having the following structure was added to a final concentration of 45 nM and 300 μM, respectively.

H ₂ O ₂ was added to a final concentration of 25 μM, and after stirring, the mixture was allowed to stand at room temperature for 1 hour. _{NMeLuminol-N 3} unbound to rituximab was removed with a Sephadex-G25 column.

_{DBCO-PEG 4-} TAMRA (Aldrich) was added to rituximab modified with N ₃ groups to a final concentration of 10 μM, and the mixture was reacted at 37 ° C. for 30 minutes. Small molecule compounds were removed with a Sephadex-G25 column.

The final concentration of rituximab was adjusted to 50 nM, and the fluorescence in the presence and absence of the denaturant was measured with a spectrofluorescence meter (JASCO FP-8300) (Fig. 1). Guanidine was used as the denaturant and the concentration was adjusted to 7 M. _{For comparison, the fluorescence of DBCO-PEG 4-} TAMRA not bound to rituximab was also measured (Fig. 2).

As shown in the figure, the fluorescence intensity of _{DBCO-PEG 4-TAMRA was reduced by binding to rituximab.} On the other hand, _{the fluorescence intensity of DBCO-PEG 4-} TAMRA bound to rituximab was increased by the addition of a denaturant.

[Example 2] Release of quenching by lysate of cells expressing CD20 _{Rituximab bound to DBCO-PEG 4-} TAMRA was prepared in the same manner as in Example 1. The final concentration of rituximab was set to 50 nM, and the fluorescence at each concentration in the presence of cell lysate was measured with a spectrofluorescence meter (JASCO FP-8300). The cell lysates include the lysate of SU-DHL-4 cells (ATCC), which is a cell expressing an antigen (CD20) (Fig. 3, left), and the lysate of SK-BR-3 cells (ATCC), which is a cell that does not express an antigen. (Fig. 3, right) was used.

As shown in the figure, when SK-BR-3 cell lysate was added, the fluorescence intensity hardly increased, whereas when SU-DHL-4 cell lysate was added, the fluorescence intensity was the concentration of lysate. Increased depending on.

[Example 3] Effect of linker on quenching cancellation
DBCO-TAMRA was used instead of DBCO-PEG ₄ -TAMRA, and the others were the same as in Example 2 with SU-DHL-4 cell lysate (Fig. 4 left) or SK-BR-3 cell lysate (Fig. 4 right). ) Fluorescence in the presence was measured with a spectroscopic fluorometer (JASCO FP-8300). DBCO-TAMRA has been removed is PEG ₄ as a linker DBCO-PEG ₄ -TAMRA, DBCO and TAMRA are close.

Comparing FIGS. 3 and 4, DBCO-TAMRA has a larger increase in fluorescence intensity when lysate is added to antigen-expressing cells, and a shorter linker between DBCO and TAMRA is more suitable for antigen detection. It is thought that there is.

[Example 3] Preparation of modified antibody to which various fluorescent dyes are bound
A 100 mM DMF solution of DBCO-amine (Aldrich) (2 eq), N, N-diisopropylethylamine (2 eq) and an NHS-bound fluorescent dye (1 eq) were reacted in DMF for 1 hour to give the reaction mixture DBCO. as bound fluorophore (1 eq) to a final concentration of 10 [mu] M, in addition to trastuzumab modified with modified rituximab or N ₃ group in N ₃ group, it was reacted for 30 minutes at 37 ° C.. Small molecule compounds were removed with a Sephadex-G25 column. Trastuzumab modified with N ₃ group was prepared in the same manner as rituximab modified with N ₃ group (Example 1).

The fluorescent dyes used in the study were Rhodamine dyes including TAMRA, Fluorescein dyes, Carbopyronin dyes, Coumin dyes, NBD dyes, Acridine dyes, and BODIPY dyes, for a total of 20 types (Fig. 5). For antibodies modified with these fluorescent dyes, the fluorescence intensity in the quenching state (Quenching) and the fluorescence intensity in the dequenching state (De-Quenching) are determined, and the ratio of the two (De-Quenching intensity / Quenching intensity) is the response ratio (Res). . Ratio). The response ratio of the antibody modified with the above fluorescent dye is shown in the table below. The dequenched state was generated not by the addition of the antigen but by the addition of the denaturing agent.

When using Rituximab, Rhodamine dye TAMRA, ATTO 488, ATTO 520, ATTO565, ATTO 590, Carbopyronin dye ATTO 610, Coumarin dye ATTO 425, Acridine dye ATTO 495, BODIPY dye BODIPY FL, BODIPY R6G, BODIPY 558, BODIPY TMR and BODIPY 581 showed good response ratios. On the other hand, when Trastuzumab was used, Rhodamine dye ATTO 520, Carbopyronin dye ATTO 610, Coumarin dye ATTO 425, BODIPY dye BODIPY FL, BODIPY R6G, BODIPY 558, BODIPY TMR, and BODIPY 581 showed good response ratios. rice field.

[Example 4] Method for producing electrochemically modified rituximab Rituximab (Chugai Pharmaceutical Co., Ltd.), which is an anti-human CD20 antibody, was dissolved in Tris Buffer (50 mM Tris-HCl, pH 7.4) at a concentration of 5 μM, and NMeLuminol-N ₃ Was added to a concentration of 300 μM. This was performed with an electrochemical reactor (positive electrode, counter electrode: RVC electrode (IKA), reference electrode: Ag / AgCl (IKA)) as shown in FIG. The voltage was applied by HSV-110 (Hokuto Denko) at a stirring speed of 400 rpm. After applying a constant voltage of 0.7 V for 30 minutes, the small molecule compounds were removed with a Sephadex-G25 column.

DBCO-TAMRA was added as a fluorescent dye to a final concentration of 10 μM with respect to 5 μM of rituximab modified with N _{3 groups, and the mixture was reacted at 37 ° C. for 30 minutes.} Small molecule compounds were removed with a Sephadex-G25 column.

The fluorescence spectra of the TAMRA-labeled rituximab thus prepared in the quenching state (in the absence of the denaturant) and in the dequenching state (in the presence of the denaturing agent) are shown in FIG. As shown in FIG. 7, fluorescently labeled rituximab produced by an electrochemical method is dequenched by the addition of a modifier, similar to fluorescently labeled rituximab produced using _{HRP and H 2} O _2. woke up.

All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

Since the fluorescently labeled antibody or antibody fragment of the present invention can be used as a diagnostic agent for various diseases, it can be used in the industrial field related to the production of such a diagnostic agent.

Claims (18)

An antibody or antibody fragment labeled with a fluorescent dye, wherein the fluorescent dye has the following properties (1) and (2).
(1) In the absence of the antigen, the fluorescent dye is quenched, and in the presence of the antigen, the quenching of the fluorescent dye is canceled.
(2) The fluorescent dye is contained in a compound that binds to an amino acid residue in an antibody or antibody fragment. Compounds that bind to amino acid residues in an antibody or antibody fragment are represented by the following general formulas (I) to (VIII).

[In the formula, A represents a conjugated ring, L represents a linker with a fluorescent dye at the end (however, if L is on A, L may be at any position on A), R. ¹ represents a hydrogen atom or represents one or two amino, acetamide, hydroxy, alkyl, or alkoxy groups present at any position on A, R ² , R ³ , R ⁴ , And R ⁵ represent an aromatic group which may have a hydrogen atom, an alkyl group and a substituent, respectively. ]
The fluorescently labeled antibody or antibody fragment according to claim 1, wherein the compound is represented by. The fluorescently labeled antibody or antibody fragment according to claim 1, wherein the compound that binds to an amino acid residue in the antibody or antibody fragment is a compound that binds to a tyrosine residue in the antibody or antibody fragment. Compounds that bind to tyrosine residues in an antibody or antibody fragment are represented by the following general formulas (I) to (VI).

[In the formula, A represents a conjugated ring, L represents a linker with a fluorescent dye at the end (however, if L is on A, L may be at any position on A), R. ¹ represents a hydrogen atom, or one or two amino groups, acetamide groups, hydroxy groups, alkyl groups, or alkoxy groups present at arbitrary positions on A, and R ² and R ³ represent, respectively. Represents an aromatic group that may have a hydrogen atom, an alkyl group, or a substituent. ]
The fluorescently labeled antibody or antibody fragment according to claim 3, wherein the compound is represented by. The compound that binds to the tyrosine residue in the antibody or antibody fragment is the following general formula (Ia).

[In the formula, L represents a linker that exists at an arbitrary position on the benzene ring and has a fluorescent dye at the end. ]
The fluorescently labeled antibody or antibody fragment according to claim 3, wherein the compound is represented by. The invention according to any one of claims 1 to 5, wherein the fluorescent dye is a fluorescent dye having 4,4-difluoro-4-bora-3a, 4a-diaza-s-indacen as a basic skeleton. Fluorescently labeled antibody or antibody fragment. The fluorescently labeled antibody according to any one of claims 1 to 6, wherein the antibody or antibody fragment comprises a light chain and a heavy chain, and the fluorescent dye is bound to a variable region of the light chain or the heavy chain. Or an antibody fragment. The claim is characterized in that the antibody or antibody fragment is bound to a quencher for the fluorescent dye, and the quencher is bound to a variable region of a chain other than the chain to which the fluorescent dye is bound. Item 7. The fluorescently labeled antibody or antibody fragment according to Item 7. The antibody or antibody fragment is bound to another fluorescent dye that forms a FRET pair with the fluorescent dye, and the other fluorescent dye is in a variable region of a chain different from the chain to which the fluorescent dye is bound. The fluorescently labeled antibody or antibody fragment according to claim 7, which is bound. The fluorescently labeled antibody or antibody fragment according to any one of claims 1 to 9, wherein the antibody or antibody fragment is an antibody or antibody fragment against a substance that serves as a marker for a disease. A diagnostic agent for a disease, which comprises the fluorescently labeled antibody or antibody fragment according to claim 10. A method for producing a fluorescently labeled antibody or antibody fragment, which comprises the following steps (1) and (2).
(1) A compound that binds to an amino acid residue in an antibody or antibody fragment and contains a functional group used in a click reaction is reacted with the antibody or antibody fragment to form an amino acid residue in the antibody or antibody fragment. The process of combining,
(2) A step of reacting a fluorescent dye with a functional group used in a click reaction in a compound that binds to an amino acid residue in an antibody or antibody fragment, and binding to this functional group. The compounds that bind to the amino acid residues in the antibody or antibody fragment are the following general formulas (Ib) to (VIIIb).

[In the formula, A represents a conjugated ring and Lb represents a linker having a functional group used for the click reaction at the terminal (however, if Lb is present on A, Lb is present at an arbitrary position on A). Good), R ¹ represents a hydrogen atom, or represents one or two amino, acetamide, hydroxy, alkyl, or alkoxy groups present at any position on A, R ² , R ³ , R ⁴ , and R ⁵ represent aromatic groups that may have hydrogen atoms, alkyl groups, and substituents, respectively. ]
The method for producing a fluorescently labeled antibody or antibody fragment according to claim 12, wherein the compound is represented by. The method for producing a fluorescently labeled antibody or antibody fragment according to claim 12, wherein the compound that binds to an amino acid residue in the antibody or antibody fragment is a compound that binds to a tyrosine residue in the antibody or antibody fragment. .. Compounds that bind to tyrosine residues in an antibody or antibody fragment are represented by the following general formulas (Ib) to (VIb).

[In the formula, A represents a conjugated ring and Lb represents a linker having a functional group used for the click reaction at the terminal (however, if Lb is present on A, Lb is present at an arbitrary position on A). Good), R ¹ represents a hydrogen atom, or represents one or two amino, acetamide, hydroxy, alkyl, or alkoxy groups present at any position on A, R ² and R ³ represents an aromatic group which may have a hydrogen atom, an alkyl group and a substituent, respectively. ]
The method for producing a fluorescently labeled antibody or antibody fragment according to claim 14, wherein the compound is represented by. A compound that binds to a tyrosine residue in an antibody or antibody fragment has the following general formula (Iab).

[In the formula, Lb represents a linker that exists at an arbitrary position on the benzene ring and has a functional group used in the click reaction at the end. ]
The method for producing a fluorescently labeled antibody or antibody fragment according to claim 14, wherein the compound is represented by. The invention according to any one of claims 12 to 16, wherein the fluorescent dye is a fluorescent dye having 4,4-difluoro-4-bora-3a, 4a-diaza-s-indacen as a basic skeleton. A method for producing a fluorescently labeled antibody or antibody fragment.

Any of claims 12 to 17, wherein step (1) is a step of applying a voltage to a solution containing an antibody or an antibody fragment and a compound that binds to an amino acid residue in the antibody or antibody fragment. The method for producing a fluorescently labeled antibody or antibody fragment according to item 1.

Images